The SNARE hypothesis states that the folding and assembly of four-helix SNARE complex bundles drives intracellular membrane fusion, including fusion in exocytosis of synaptic vesicles at the plasma membranes of synaptic terminals in neurons. The neuronal SNAREs are syntaxin 1 a and SNAP-25 on the plasma membrane and synaptobrevin 2 on the vesicle membrane. Syntaxin contributes one and SNAP-25 contributes two helices to the active acceptor SNARE complex on the target membrane. Synaptic vesicles dock to the presynaptic plasma membrane by contributing synaptobrevin to the nascent SNARE complex. How far this trans-SNARE complex that bridges the two membranes is folded is presently not known. The trans-SNARE complex is then thought to zipper-fold from the N- towards the C-terminus of the parallel fourhelix bundle. The bundle, thereby pulling the two membranes into close proximity. In a final step, SNARE complex folding proceeds into the transmembrane domains of syntaxin and synaptobrevin to form a cis- SNARE complex, i.e. a step which must be coupled with merging the lipid bilayers of the two membranes. The research proposed in this project will dissect the described folding steps and correlate them with different stages of vesicle docking and membrane fusion. Magnetic resonance approaches will be taken to determine (dynamic) structures of SNARE folding intermediates and micro-fluorescence approaches will be taken to determine the kinetics and higher architecture of the evolving fusion pore in artificial and cellular membrane preparations.